Considering the importance of the progesterone (P)-proliferative signal to mammary development and tumorigenesis, our long-term goal is to exploit experimental mouse genetics to gain a more mechanistic understanding of P's role as an endocrine mammogen, in vivo. Toward this end, our recent studies revealed that in the proliferating murine mammary gland, the majority of PR positive (+) cells were surprisingly nonproliferative but were in close association with a subgroup of PR negative (-) cells which proliferate in response to P; a similar cellular organization pattern for PR expression occurs in the human and rat mammary gland. Importantly, derailment of this distinct patterning for mammary PR expression is linked to a number of aberrant mammary growth phenotypes, including neoplasia. The foregoing observations strongly support our hypothesis of an evolutionary conserved cellular mechanism for P-action in the normal gland in which PR+ cells receive, transduce, and then relay (via a paracrine pathway) the P-proliferative signal to neighboring PR- cells, which are then directed toward a pathway of proliferation. Our hypothesis predicts the existence of an intraepithelial paracrine molecular pathway(s), which mediates the P-proliferative signal; the Wnt-4 pathway has been implicated as such a pathway. To test our hypothesis, Specific Aim 1 will employ our PR-LacZ reporter mouse, a recently generated PR-enhanced green fluorescent protein knockin mouse, fluorescence-activated cell sorting, and mammary epithelial transplantation approaches to query-at the cellular level-the functional importance of P's proposed paracrine mechanism of action in the normal mammary gland. Specific Aim 2 will define the developmental importance of PR expression during early mammary development to PR's function in the adult gland by using the tetracycline regulated expression system (the TET-ON system) to temporally control PR expression in the mammary epithelium of the PR knockout (PRKO) mouse. Using the TET-ON system and the PRKO, Specific Aim 3 will address whether Wnt-4 is a bona-fide paracrine mediator of the P-proliferative signal in the murine mammary gland. Finally, Specific Aim 4 will employ comparative transcriptional profiling of normal and PRKO mammary glands to identify the complete spectrum of paracrine mediators of the P-mammary signal. Application of microarray approaches to murine mammary tumor models (developed during the last grant period) will also uncover signature gene-networks specific for either hormone-dependent or -independent breast tumors; this aim should also furnish important molecular targets for future breast cancer diagnosis, prognosis and/or therapy. [unreadable] [unreadable]